As far as known, prior workers, such as Mashberg (U.S. Pat. No. 4,321,251), who instigated the use of TC for in vivo identification of dysplasia, used prior art dye products in which the conformational isomers of TC plus the N-demethylation and N,N-demethylation derivatives of the conformational isomers were less than 80% of the dye composition and in which the two N-demethylation derivatives of the conformational isomers, formed greater than about 20% % of the dye composition. According to my information, prior workers were unaware of the exact composition of their “TC” products and manufacturers of prior art TC products were unable to reproducibly prepare them. In fact, the prevalent literature description of the quality of TC was simply that it have “good color value”. The Biological Stain Commission specifies an analytical titration procedure for determining only the “organic dye content” of the TC material. The prior art use of such loosely defined “TC” resulted in anomalous clinical observations and serious problems in obtaining necessary regulatory clearances to manufacture and market such products for use in human diagnostic procedures.
In addition to the problem of variable initial composition, prior art TC and other thiazine biological stains were subject to time-related variations in composition. For example, Dean et al. in “Stains Technology”, Vol. 32, No. 1, pp. 35 et seq. (1977) recommended that thiazine dyes in methanolic solutions should be refrigerated to prevent further oxidative N-demethylation. Liao, et al. reported in “Stains Technology”, Vol. 57, No. 1, pp. 23 et seq. (1982) that reduction in methylene blue content by precipitation from methanolic Wright's stain solution could be markedly decreased by simultaneous addition of diethylamine hydrochloride and dimethylamine hydrochloride.
In my U.S. Pat. No. 6,086,852, I describe a process for reproducibly manufacturing high-quality TC products initially having a high proportion of the conformational isomers with respect to the N-demethylation products of such isomers. While exclusion of contact with air and avoidance of high temperatures retard oxidative N-demethylation of the conformational isomers, it is observed that, in the absence of free radical scavengers, such as metal ions, alcoholic solutions of this TC product undergo light-induced or photochemical N-demethylation.